Extracellular Nucleotides Activate non‐Selective Cation and Ca2+‐Dependent K+ Channels in Rat Osteoclasts

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Abstract
1 Extracellular ATP elevates cytosolic free Ca2+ concentration ([Ca2+]i) in osteoclasts, but its effects on ion channels have not been reported previously. Membrane currents and [Ca2+]i were recorded in isolated rat osteoclasts using patch clamp and fluorescence techniques. 2 At negative membrane potentials, ATP (1–100 μm) activated an inward current that peaked rapidly and then declined. A later current was outward at potentials positive to the equilibrium potential for K+ (EK) and showed oscillations. 3 The initial inward current, studied in isolation using Cs+ in the electrode solution, showed rapid activation, inward rectification and reversal at +3 ± 4 mV. Reduction of [Na+]o to 10 mM shifted the reversal potential to –21 ± 3 mV, indicating that ATP activates a non‐selective cation current, consistent with involvement of P2X receptors. 4 The later current activated by ATP, studied with K+ in the electrode solution, exhibited a linear I–V relationship, and reversed at –71 ± 4 mV. The reversal potential shifted 51 mV per 10‐fold change of [K+]o, indicating that ATP activates a K+ current (IK). 5 In fura‐2‐loaded cells, ATP caused elevation of [Ca2+]i that persisted in Ca2+‐free solution, indicating that ATP induced release of Ca2+ from intracellular stores, consistent with involvement of P2Y receptors. Simultaneous patch clamp and fluorescence recordings revealed that IK was associated with the elevation of [Ca2+]i. Using a Ca2+ ionophore (4Br‐A23l87) to elevate [Ca2+]iIK activated when [Ca2+]i exceeded ∼400 nm, with half‐maximal activation at 580 ± 50 nM. 6 In cell‐attached patches, ATP activated a channel with a conductance of 48 ± 6 pS, that reversed direction near EK Channel open probability increased with elevation of [Ca2+]i, indicating the Ca2+ dependence of this channel. 7 These results demonstrate that rat osteoclasts express two types of purinoceptors. P2X receptors give rise to non‐selective cation current. P2Y receptors mediate Ca2+ release from stores, causing activation of a Ca2+‐dependent K+ channel.